Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.

Deciphering the interaction of bacteria inoculants with the recipient endophytic community in grapevine micropropagated plants.

Engineering the plant microbiome with beneficial endophytic bacteria can improve the growth, health, and productivity of the holobiont. Here, we administered two beneficial bacterial strains, Kosakonia VR04 sp. and Rhizobium GR12 sp., to micropropagated grapevine cuttings obtained via somatic embryogenesis. While both strains colonized the plant endosphere, only Rhizobium GR12 sp. increased root biomass under nutritional-deficit conditions, as supported by the plant growth promotion traits detected in its genome. Phylogenetic and co-occurrence analyses revealed that the plant native bacterial community, originally dominated by Streptococcaceae and Micrococcaceae, dramatically changed depending on the inoculation treatments, as invading strains differently affected the relative abundance and the interactions of pre-existing taxa. After 30 days of plantlets' growth, Pantoea became a predominant taxon, and considering untreated plantlets as references, Rhizobium sp. GR12 showed a minor impact on the endophytic bacterial community. On the other hand, Kosakonia sp. VR04 caused a major change in community composition, suggesting an opportunistic colonization pattern. Overall, the results corroborate the importance of preserving the native endophytic community structure and functions during plant microbiome engineering.IMPORTANCEA better comprehension of bacterial colonization processes and outcomes could benefit the use of plant probiotics in the field. In this study, we applied two different beneficial bacteria to grapevine micropropagated plantlets and described how the inoculation of these strains impacts endophytic microbiota assembly. We showed that under nutritional deficit conditions, the response of the receiving endophytic bacterial communities to the invasion of the beneficial strains related to the manifestation of plant growth promotion effects by the inoculated invading strains. Rhizobium sp. GR12 was able to preserve the native microbiome structure despite its effective colonization, highlighting the importance of the plant-endophyte associations for the holobiont performance. Moreover, our approach showed that the use of micropropagated plantlets could be a valuable strategy to study the interplay among the plant, its native microbiota, and the invader on a wider portfolio of species besides model plants, facilitating the application of new knowledge in agriculture.

CRISPR/Cas9 genome-editing applied to MdPGT1 in apple results in reduced foliar phloridzin without impacting plant growth.

Phloridzin is the most abundant polyphenolic compound in apple (Malus × domestica Borkh.), which results from the action of a key phloretin-specific UDP-2'-O-glucosyltransferase (MdPGT1). Here, we simultaneously assessed the effects of targeting MdPGT1 by conventional transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing. To this end, we conducted transcriptomic and metabolic analyses of MdPGT1 RNA interference knockdown and genome-edited lines. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology, whereas genome-edited lines exhibited normal growth despite reduced foliar phloridzin. RNA-sequencing analysis identified a common core of regulated genes, involved in phenylpropanoid and flavonoid pathways. However, we identified genes and processes differentially modulated in stunted and genome-edited lines, including key transcription factors and genes involved in phytohormone signalling. Therefore, we conducted a phytohormone profiling to obtain insight into their role in the phenotypes observed. We found that salicylic and jasmonic acid were increased in dwarf lines, whereas auxin and ABA showed no correlation with the growth phenotype. Furthermore, bioactive brassinosteroids were commonly up-regulated, whereas gibberellin GA4 was distinctively altered, showing a sharp decrease in RNA interference knockdown lines. Expression analysis by reverse transcriptase-quantitative polymerase chain reaction expression analysis further confirmed transcriptional regulation of key factors involved in brassinosteroid and gibberellin interaction. These findings suggest that a differential modulation of phytohormones may be involved in the contrasting effects on growth following phloridzin reduction. The present study also illustrates how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple.

Unlocking grapevine in vitro regeneration: Issues and perspectives for genetic improvement and functional genomic studies.

In vitro plant regeneration is a pivotal process in genetic engineering to obtain large numbers of transgenic, cisgenic and gene edited plants in the frame of functional gene or genetic improvement studies. However, several issues emerge as regeneration is not universally possible across the plant kingdom and many variables must be considered. In grapevine (Vitis spp.), as in other woody and fruit tree species, the regeneration process is impaired by a recalcitrance that depends on numerous factors such as genotype and explant-dependent responses. This is one of the major obstacles in developing gene editing approaches and functional genome studies in grapevine and it is therefore crucial to understand how to achieve efficient regeneration across different genotypes. Further issues that emerge in regeneration need to be addressed, such as somaclonal mutations which do not allow the regeneration of individuals identical to the original mother plant, an essential factor for commercial use of the improved grapevines obtained through the New Breeding Techniques. Over the years, the evolution of protocols to achieve plant regeneration has relied mainly on optimizing protocols for genotypes of interest whilst nowadays with new genomic data available there is an emerging opportunity to have a clearer picture of its molecular regulation. The goal of this review is to discuss the latest information available about different aspects of grapevine in vitro regeneration, to address the main factors that can impair the efficiency of the plant regeneration process and cause post-regeneration problems and to propose strategies for investigating and solving them.

Raman Spectroscopy Applications in Grapevine: Metabolic Analysis of Plants Infected by Two Different Viruses.

Grapevine is one of the most cultivated fruit plant among economically relevant species in the world. It is vegetatively propagated and can be attacked by more than 80 viruses with possible detrimental effects on crop yield and wine quality. Preventive measures relying on extensive and robust diagnosis are fundamental to guarantee the use of virus-free grapevine plants and to manage its diseases. New phenotyping techniques for non-invasive identification of biochemical changes occurring during virus infection can be used for rapid diagnostic purposes. Here, we have investigated the potential of Raman spectroscopy (RS) to identify the presence of two different viruses, grapevine fan leaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in Vitis vinifera cv. Chardonnay. We showed that RS can discriminate healthy plants from those infected by each of the two viruses, even in the absence of visible symptoms, with accuracy up to 100% and 80% for GFLV and GRSPaV, respectively. Chemometric analyses of the Raman spectra followed by chemical measurements showed that RS could probe a decrease in the carotenoid content in infected leaves, more profoundly altered by GFLV infection. Transcriptional analysis of genes involved in the carotenoid pathway confirmed that this biosynthetic process is altered during infection. These results indicate that RS is a cutting-edge alternative for a real-time dynamic monitoring of pathogens in grapevine plants and can be useful for studying the metabolic changes ensuing from plant stresses.

Grapevine virome and production of healthy plants by somatic embryogenesis.

Grapevine (Vitis spp.) is a widespread fruit tree hosting many viral entities that interact with the plant modifying its responses to the environment. The production of virus-free plants is becoming increasingly crucial for the use of grapevine as a model species in different studies. Using high-throughput RNA sequencing, the viromes of seven mother plants grown in a germplasm collection vineyard were sequenced. In addition to the viruses and viroids already detected in grapevine, we identified 13 putative new mycoviruses. The different spread among grapevine tissues collected in vineyard, greenhouse and in vitro conditions suggested a clear distinction between viruses/viroids and mycoviruses that can successfully be exploited for their identification. Mycoviruses were absent in in vitro cultures, while plant viruses and viroids were particularly accumulated in these plantlets. Somatic embryogenesis applied to the seven mother plants was effective in the elimination of the complete virome, including mycoviruses. However, different sanitization efficiencies for viroids and grapevine pinot gris virus were observed among genotypes. The absence of mycoviruses in in vitro plantlets, associated with the absence of all viral entities in somaclones, suggested that this regeneration technique is also effective to eradicate endophytic/epiphytic fungi, resulting in gnotobiotic or pseudo-gnotobiotic plants.